Protein lattice

ABSTRACT

Protein lattice ( 1 ) having a regular structure with a repeating unit repeating in three dimensions may have many uses, for example to support an array of macromolecular entities for x-ray crystallography. The repeating unit comprises protein protomers ( 2 ) which each comprise at least two monomers ( 5, 6 ) fused together. The monomers ( 5, 6 ) are each monomers of a respective oligomer assembly ( 3, 4 ) into which the monomers are assembled for assembly of the protomers into the lattice. The first oligomer assembly ( 3 ) has a set of rotational symmetry axes extending in three dimensions. In said protomers ( 2 ), further monomers ( 6 ) fused to said first monomers ( 5 ) are monomers of respective further oligomer assemblies ( 4 ) which have a rotational symmetry axis of the same order as a respective one of said set of rotational symmetry axes of said first oligomer assembly ( 3 ). Thus, the repeating unit includes protomers ( 2 ) with the first monomers ( 5 ) of the protomers ( 2 ) being assembled into said first oligomer assembly ( 3 ) and, in respect of respective ones of said set of rotational symmetry axes, with further monomers ( 6 ) of the protomers ( 2 ) fused to respective first monomers ( 3 ) being assembled into respective further oligomer assemblies ( 4 ). As a result of the symmetry of the oligomer assemblies ( 3, 4 ) said rotational symmetry axis of said respective further oligomer assemblies ( 4 ) is aligned with the respective rotational symmetry axis of said first oligomer assembly ( 3 ). Thus, an N-fold fusion between the oligomer assemblies ( 3, 4 ) is produced and the rotational symmetry axes of the oligomer assemblies ( 3, 4 ) define the symmetry of the lattice.

The present invention relates to protein lattices having a regularstructure repeating in three dimensions. The protein lattices are basedon symmetrical oligomer assemblies capable of self-assembly from themonomers of the oligomer assembly. Such protein lattices may have poreswith dimensions of the order of nanometres to hundreds of nanometres. Assuch, the protein lattices are nanostructures which have many potentialuses, for example as a matrix to support macromolecular entities forX-ray crystallography.

WO-00/68248 discloses regular protein structures based on symmetricaloligomer assemblies capable of self-assembly. In particular, WO-00/68248discloses structures formed from protein protomers (referred to as a“fusion protein” in WO-00/68248) comprising at least two monomers(referred to as “oligomerization domains” in WO-00/68248) which are eachmonomers of a respective symmetrical oligomer assembly. Self-assembly ofthe monomers into the oligomer assembly causes assembly of the regularstructures themselves. Several different types of structures aredisclosed, including discrete structures and structures extending inone, two and three dimensions.

In WO-00/68248, the relative orientations of the monomers within theprotomers are selected to provide the desired regular structure uponself-assembly. The monomers are fused together through a rigid linkinggroup which is carefully selected to provide the requisite relativeorientation of the monomers in the protomer. For example, in thelaboratory production reported in WO-00/68248, the selection of theprotomer was performed using a computer program to model monomersconnected by a linking group in the form of a continuous, interveningalpha-helical segment over a range of incrementally increased lengths.Thus, the lattices suggested in WO-00/68248 having a regular structurerepeating in three dimensions are formed from protomers comprising twomonomers of respective dimeric or trimeric oligomer assemblies which aresymmetrical about a single rotational axis. The relative orientation ofthe two monomers is selected to provide a specific angle of intersectionbetween the rotational symmetry axis of the two oligomer assemblies.Thus, there is a single fusion between the two oligomer assemblies andthe relative orientation of the oligomer assemblies is controlled bycareful selection of the linking group providing the fusion.

WO-00/68248 only reports laboratory production of protein structures ofa discrete cage and a filament extending in one dimension. It isexpected that application of the teaching of WO-00/68248 to proteinlattices repeating in three dimensions would encounter the followingdifficulties. Firstly, it is expected that there would be a difficultyin design arising from the requirement to select the relativeorientation of the monomers within the protomer appropriate forconstructing a lattice. This would probably reduce the numbers of typesof oligomer assembly available to form a protein lattice, and hence makeit difficult to identify suitable proteins. Secondly, it is expectedthat practical difficulties would be encountered during assembly. Thestructures disclosed in WO-00/68248 rely on the rigidity of the fusionbetween monomers in protomers which forms the single fusion betweenoligomer assemblies. WO-00/68248 teaches that the relative orientationof the monomers in the protomers controls the relative orientation ofthe oligomer assemblies in the resultant structure, so it is expectedthat flexing of the fusion away from the desired relative orientationwould reduce the reliability of self-assembly. It is expected that sucha problem would become more acute as the size of the repeating unitincreases, thereby providing a practical restriction on the reliableproduction of lattices with a relatively large pore sizes.

Accordingly, it would be desirable to provide protein lattices having adifferent type of structure in which these expected problems might bealleviated.

According to a first aspect of a present invention, there is provided aprotein lattice having a regular structure with a repeating unitrepeating in three dimensions, the repeating unit comprising proteinprotomers which each comprise at least two monomers fused together, themonomers each being monomers of a respective oligomer assembly intowhich the monomers are assembled for assembly of the protomers into thelattice, wherein the repeating unit comprises protomers comprising atleast a first monomer which is a monomer of a first oligomer assemblywhich has a quaternary structure which is symmetrical in threedimensions.

As a result of using at least a first oligomer assembly which issymmetrical in three dimensions, the structure of the repeating unit andhence the protein lattice is derived from the symmetry of the oligomerassembly. In particular, it is not dependent on the relative orientationof the monomers within the protomer. Therefore, protein lattices inaccordance with the present invention may be designed by selectingoligomers assemblies wherein at least the first oligomer assembly has anappropriate three dimensional symmetry to build a lattice repeating inthree dimensions. Protomers are then produced comprising monomers of theselected oligomer assemblies fused together. Subsequently, the protomersare allowed to self-assemble under suitable conditions. As described inmore detail below, the chosen symmetries of the oligomer assembliescause the protomers to self-assemble into the protein lattice.

To assist in understanding, reference is made to FIG. 1 whichillustrates a particular example of a protein lattice in accordance withthe present invention, as described in more detail below. In particular,the protein lattice 1 has a regular structure with a repeating unitcomprising a first oligomer assembly 3 which is symmetrical in threedimensions, which in this example is human heavy chain ferritin whichhas octahedral symmetry. Each of the monomers 5 of the first oligomerassembly 3 is fused to a further monomer 6 of a further oligomerassembly 4 which in this example is E. Coli PurE has symmetry belongingto the dihedral D₄ point group 4. The further monomers 6 are assembledinto the further oligomer assemblies 4 arranged with their rotationalsymmetry axes of order 4 aligned along the rotational symmetry axes oforder 4 of the first oligomer assembly 3. Thus, the symmetry of therepeating unit, and hence the symmetry of the protein lattice 1, is thesame as the symmetry of the set of rotational symmetry axes of order 4,as will be described in more detail below.

Accordingly, the present invention involves the use of a different classof oligomers assemblies from that used in WO-00/68248 and provides thebenefit that one is not restricted by the selection of the relativeorientation of the monomers within the protomer. Thus it is expectedthat the design of protein lattice will be assisted in that the relativeorientation of the monomers withing the protomer is a less criticalconstraint. Similarly, it is expected that more reliable assembly of theprotein lattices will be possible, as described in more detail below.

According to other aspects of the present invention, there is providedan individual protomer or plural protomers capable of self-assembly toform such a protein lattice, as well as polynucleotides encoding suchprotomers, vectors and host cells capable of expressing such promotersand methods of making the protomers.

The present invention will now be described in more detail by way ofnon-limitative example with reference to the accompanying drawings inwhich:

FIG. 1 is a diagram schematically illustrating, for a first proteinlattice, the design of a homologous protomer based on two oligomerassemblies and production of the lattice itself;

FIG. 2 is a diagram schematically illustrating, for a second proteinlattice, the design of two heterologous protomers based on threeoligomer assemblies and production of the lattice itself; and

FIG. 3 is a picture of an experimentally produced protein lattice of thetype illustrated in FIG. 1.

Protein lattices in accordance with the present invention may bedesigned by selecting oligomer assemblies, at least a first of which issymmetrical in three dimension, which fused together produce a repeatingunit which is capable of repeating in three dimensions. As the symmetryof the repeating unit, and hence the lattice as a whole, depends on thesymmetry of the oligomer assemblies, this involves a selection ofoligomer assemblies having a quaternary structure which providesappropriate symmetries. This is a straightforward task, because thesymmetries of oligomer assemblies are generally available in thescientific literature on proteins, for example from The Protein DataBank; H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H.Weissig, I. N. Shindyalov & P. E. Boume; Nucleic Acids Research, 28 pp.235-242 (2000) which is the single worldwide archive of structure dataof biological macromolecules, also available through websites such ashttp://www.rcsb.org.

In some lattices, the repeating unit repeats in the same orientationacross the lattice. In other lattices two or more adjacent repeatingunits together form a unit cell which repeats in the same orientationacross the lattice, but with the repeating units within a unit cellarranged in different orientations.

Examples of oligomer assemblies which produce lattices with a repeatingunit repeating in three dimension are given below.

Advantageously, the first oligomer assembly has a quaternary structurewith a set of rotational symmetry axes extending in three dimensions. Asa result, said repeating unit includes protomers with the first monomersof the protomers being assembled into said first oligomer assembly and,in respect of respective ones of said set of rotational symmetry axes,with further monomers of the protomers fused to respective firstmonomers being arranged symmetrically around said respective one of saidset of rotational symmetry axes.

The arrangement of the repeating unit, and hence the lattice as a whole,is therefore dependent on the symmetries of the first oligomer assembly.In particular, in the assembled first oligomer assembly, inevitably andby definition, there are groups of first monomers arranged symmetricallyaround each of the set of rotational symmetry axes of the first oligomerassembly. This is because the symmetry results from the identicalmonomers being so arranged around the rotational symmetry axes.

Since the further monomers are each fused to a respective first monomer,it follows that groups of the further monomers are also arrangedsymmetrically around each of the set of rotational symmetry axes. Thefurther monomers are held in this symmetrical arrangement by beingattached to first monomers in the first oligomer assembly. These groupsof symmetrically arranged further monomers fused to the first oligomerassembly self-assemble with other monomers (which may be correspondingfurther monomers of another repeating unit, or may be monomers in adifferent part of the same unit cell) to form further oligomerassemblies, which are also arranged symmetrically around the set ofrotational symmetry axes of the first oligomer assembly.

Thus, the arrangement of the repeating unit, and hence the lattice as awhole, is dependent on the symmetries of the first oligomer assembly,not on the relative orientation of the monomers within an individualprotomer. In other words, the present invention provides the advantagethat the three dimensional structure of the protein lattice may be basedsolely on the symmetries of the oligomer assemblies. This providesadvantages in the design of the protein lattices. This is to say, thedesign of the repeating unit and hence the lattice as a whole may bebased on the symmetries of the oligomer assemblies. This makes it easyto select appropriate oligomer assemblies for use in the proteinlattice.

Desirably, the first oligomer assembly has a quaternary structure with aset of rotational symmetry axes extending in three dimensions, and, insaid protomers, further monomers fused to said first monomers aremonomers of respective further oligomer assemblies which have arotational symmetry axis of the same order as a respective one of saidset of rotational symmetry axes of said first oligomer assembly. As aresult, said repeating unit includes protomers with the first monomersof the protomers being assembled into said first oligomer assembly and,in respect of respective ones of said set of rotational symmetry axes,with further monomers of the protomers fused to respective firstmonomers being assembled into respective further oligomer assemblieswith said rotational symmetry axis of said respective further oligomerassemblies being aligned with the respective rotational symmetry axis ofsaid first oligomer assembly.

The arrangement of the repeating unit and hence the lattice as a wholeare therefore dependent on the symmetries of the first and furtheroligomer assemblies. In particular, as described above, in the firstoligomer assembly there are groups of first monomers arrangedsymmetrically around each of the set of rotational symmetries axes,which in turn result in groups of the further monomers fused to thefirst monomers also being arranged symmetrically around each of the setof rotational symmetry axes of the first oligomer assembly. These groupsof symmetrically arranged further monomers fused to the first oligomerassembly self-assemble with other monomers to form the further oligomerassembly. The further monomers may be further monomers of anotherrepeating unit or may be monomers in a different part of the samerepeating unit.

As a result of the further monomers fused to the first oligomer assemblybeing arranged symmetrically around a rotational symmetry axis of thefirst oligomer assembly, it follows that the further oligomer assemblyis held with the group of fused further monomers also held symmetricallyaround that rotational symmetry axis of the first oligomer assembly.However, inevitably and by definition, the further monomers alsoassemble in the further oligomer assembly in a symmetrical arrangementaround the rotational symmetry axis of the further oligomer assembly.Thus, the result of the further oligomer assembly having a rotationalsymmetry axis of the same order as one of the set of rotational symmetryaxes of the first oligomer assembly is that the first and furtheroligomer assemblies assemble with their symmetry axes aligned with oneanother. It follows from the symmetry of both oligomer assemblies thatthis is the most stable arrangement. This results in an N-fold fusionbetween the first and further oligomer assemblies, where N is a pluralnumber equal to the order of the respective rotational symmetry axis ofthe first oligomer assembly and the rotational symmetry axis of thefurther oligomer assembly. In each of the first and further oligomerassemblies, there are N monomers arranged around the rotational symmetryaxis, each of the monomers being fused within a respective protomer to amonomer of the other oligomer assembly.

Thus the set of rotational symmetry axes does not include all therotational symmetry axes of the first oligomer assembly. Rather the setcomprises the rotational symmetry axes of the first oligomer assemblywhich are of the same order as rotational symmetry axes of the furtheroligomer assembly. For example in the example of FIG. 1, the set ofrotational symmetry axes of the first oligomer assembly 3 are therotational symmetry axes of order 4, rather than those of order 3 or 2,due to the further oligomer assembly 4 having rotational symmetry axesof order 4. Further examples are given below.

The particular choice of symmetries of the first and further oligomerassemblies results, on assembly of the protomers into the lattice, inthe oligomer assemblies being built up with their rotational symmetryaxes aligned. This means that the arrangement of the repeating unit, andhence the lattice as a whole, is controlled by the symmetries of thefirst and further oligomer assemblies, not on the relative orientationof the monomers within an individual protomer. In other words, thepresent invention provides the advantage that the three dimensionalstructure of the protein lattice may be based solely on the symmetriesof the oligomer assemblies. This is advantageous in the design of theprotein lattice. By basing the three dimensional structure of therepeating unit and hence lattice as a whole, on the symmetries of theoligomer assemblies, it is easier to select appropriate oligomerassemblies to form a lattice. During design, the relative orientation ofthe monomers within an individual protomer in its unassembled formbecomes a much lower constraint than is present in, for example,WO-00/68248.

There are also expected to be advantages during self-assembly of thelattice. In particular, the formation of an N-fold fusion between twogiven oligomer assemblies results in the bond between the two oligomerassemblies being relatively rigid. This is expected to reduce relativemotion of the oligomer assemblies during the assembly process. This isexpected to assist in reliable formation of the lattice with theoligomer assemblies in the correct relative positions.

Although there are particular advantages in the use of a furtheroligomer assembly which has a rotational symmetry axis of the same orderas the rotational symmetry axes of the first oligomer assembly, this isnot essential. Alternatively, it would be possible for the furthermonomers arranged symmetrically around the rotational symmetry axes ofthe first oligomer assembly to be monomers of separate oligomerassemblies, for example of dimeric oligomer assemblies (beingheterologous or homologous). In that case, the further oligomer assemblywould effectively be replaced by a group of separate dimeric oligomerassemblies, equal in number to the order of the rotational symmetry axisof the first oligomer assembly, with the separate dimeric oligomerassemblies held around the rotational symmetry axis of the firstoligomer assembly in an arrangement which might or might not have theN-fold symmetry of the rotational symmetry axis of the first oligomerassembly.

The form and production of the protomers will now be described. Exceptthat the present invention involves protomers in which a differentchoice of monomers from WO-00/68248 are fused together, the form andproduction of the protomers per se, as well as the polynucleotideencoding the protomers, may be as the same as disclosed in WO-00/68248which is therefore incorporated herein by the reference.

The nature of the monomers themselves will now be described.

The monomers are monomers of oligomer assemblies which are capable ofself-assembly under suitable conditions to produce a protein lattice.The secondary and tertiary structure of the monomers is unimportant initself providing they assemble into a quaternary structure with therequired symmetry. However, it is advantageous if the protein is easilyexpressed and folded in an heterologous expression system (for exampleusing plasmid expression vector in E. Coli).

The monomers may be naturally occurring proteins, or may be modified bypeptide elements being absent from, substituted in, or added to anaturally occurring protein provided that the modifications do notsubstantially affect the assembly of the monomers into their respectiveoligomer assembly. Such modifications are in themselves known for anumber of different purposes which may be applied to monomers of thepresent invention. In other words, the monomer may be a homologue and/orfragment and/or fusion protein of a naturally occurring protein.

The monomer may be chemically modified, e.g. post-translationallymodified. For example, it may be glycosylated or comprise modified aminoacid residues.

The monomers are preferably fused genetically, although in principleother fusions are possible such as chemical fusions.

Although the monomers may be fused directly together, preferably themonomers are fused by a linking group of peptide or non-peptideelements. In general, linking two proteins by a linking group is knownfor other purposes and such linking groups may be applied to the presentinvention.

Another factor in the selection of appropriate oligomer assemblies isthe location and orientation of (a) the termini of the first monomerswhen arranged in the first oligomer assembly in its natural form (i.e.not fused to a further oligomer assembly) and (b) the termini of thefurther monomers when arranged in the further oligomer assembly in itsnatural form (i.e. not fused to the first oligomer assembly). Suchinformation on the arrangement of the termini in the oligomer assemblyin its natural form is generally available for oligomer assemblies, forexample from The Protein Data Bank referred to above. Ideally, thesetermini should have the same separation and orientation, because theywill be fused together in the assembled protein lattice to constitutethe N-fold fusion arranged symmetrically around a rotational symmetryaxis. That being said, it is not essential for the separation andorientation to be the same, because any difference may be accommodatedby deformation of the monomers near the N-fold fusion and/or by use of alinking group. Therefore, as a general point, oligomer assemblies shouldbe chosen in which the termini of both oligomer assemblies which are tobe fused together in an N-fold fusion allows formation of the fusionwithout preventing assembly of the oligomer assemblies and hence theprotein lattice.

Considering the deformation of the monomers near the N-fold fusionmentioned above, it is desirable to minimise such deformation which willtend to reduce the reliability of the assembly process. However, if alinking group is fused between the monomers, such deformation may betaken up, at least partially, by the linking group itself. This reducesthe deformation of the monomers, thereby increasing the reliability ofself-assembly because the linking group does not take part in theassembly process as regards to not being part of the naturally occurringprotein. There is a particular advantage of the use of a linking group.

Furthermore, the linking group may be specifically designed to beoriented relative to the first and further monomers in the protomer inits normal form, prior to assembly, to reduce such differences in theposition and/or orientation of the termini of the first and furthermonomers. Using position and orientation of the termini of the first andfurther monomers in the first and further oligomer assemblies in theirnatural form which is generally available for oligomer assemblies, asdiscussed above, it is possible to design an appropriate linking groupusing conventional modelling techniques.

Typically, the monomers are fused at their end termini. Alternatively,the monomers may be fused at an alternative location in the polypeptidechain so long as the native fold and symmetry of the naturally occurringoligomer assembly remains the same. For example, one of the monomers maybe inserted into a structurally tolerant portion of the other monomer,for example in a loop extending out of the oligomer assembly. Also,truncation of a monomer is feasible and may be estimated by structuralexamination.

Some examples of symmetries for the oligomer assemblies to produce aprotein lattice are as follows.

In the examples, the first oligomer assembly which is symmetrical inthree dimensions belongs to one of a tetrahedral point group, anoctahedral point group or a dihedral point group.

In some classes of protein lattice, the protomers are homologous withrespect to the monomers, ie there is a single type of protomer withinthe protein lattice. For example, Table 1 represents some simplehomologous protomers capable of forming a protein lattice. TABLE 1Homologous Protomers Protomer Class Name M N p₃p₃ Platonic 12 3 p₄p₄Platonic 24 4 p₄p₃ Platonic 24 3 (or 12) p₃d₃ Mixed 12 3 p₃d₂ Mixed 12 2p₄d₄ Mixed 24 4 p₄d₃ Mixed 24 3 p₄d₂ Mixed 24 2 d₃d₃d₂ Dihedral 6 3, 2d4d₄d₂ Dihedral 8 4, 2 d₆d₆d₂ Dihedral 12 6, 2

In Table 1, each protomer is identified by letters which represent therespective monomers of the protomer. In particular the letters identifythe point group to which the oligomer assembly of that monomer belongs.For each letter, the subscript number represents the order of the pointgroup. The letter p represents a platonic point group, so p₃ representsa tetrahedral point group, and p₄ represents an octahedral point group.The letter d represents a dihedral point group.

In the final two columns of the table, there is given the number M offirst monomers in the first oligomer assembly and the order(s) N of theset of rotational symmetry axes of the first oligomer assembly. N isalso the order of the rotational symmetry axis of the further oligomerassembly aligned with a respective rotational symmetry axis of the firstoligomer assembly, and around which there is formed an N-fold fusionbetween the first and further oligomer assemblies.

The protomers have been divided into classes which have been namedaccording to the nature of the monomers of the proteins for ease ofreference.

In both the platonic and mixed classes, the first oligomer assemblybelongs to a platonic point group, which is either a tetrahedral pointgroup or an octahedral point group.

In the mixed class, the further monomer is a monomer of an oligomerassembly belonging to a dihedral point group. In each case, the order Nof the dihedral point group, which is the order of the principalrotational symmetry axis of the dihedral point group, is equal to theorder of one of the rotational symmetry axes of the first oligomerassembly. This may either be the principal rotational symmetry axis ofthe first oligomer assembly or one of the rotational symmetry axes ofthe first oligomer assembly of lower order. The rotational symmetry axesof the first oligomer assembly of order N therefore constitute the setof rotational symmetry axes of the first oligomer assembly. Thesymmetries of the first and further oligomer assemblies results in theformation of a unit cell in which the principal rotational symmetry axisof each further oligomer assembly belonging to a dihedral point group isaligned with one of set of rotational symmetry axes of order N of theplatonic point group, with an N-fold fusion therebetween, in the mannerdescribed above.

The protein lattices of the mixed class are the easiest to visualise. Inparticular, the first oligomer assembly belonging to a platonic pointgroup may be visualised as a node from which the set of rotationalsymmetry axes of order N extend outwardly. The dihedral point groups maybe visualised as linear links with the principal rotational symmetryaxis of the dihedral point group aligned with one of the set ofrotational symmetry axes of order N of the first oligomer assembly. Inthis way, it is easy to visualise the formation of the lattice withpores in the spaces between the oligomer assemblies.

FIG. 1 illustrates a particular example of a protein lattice 1 belongingto the mixed class, in particular having a protomer 2 represented byp₄d₄. The first oligomer assembly 3 is human ferritin heavy chain (HFH)which belongs to an octahedral point group. The further oligomerassembly is E. Coli PurE which belongs to a dihedral D₄ point group oforder 4. The protomer comprises a first monomer 5 of the first oligomerassembly 3 and a further monomer 6 of the further oligomer assembly 4fused together. On assembly, the protomers 2 form a lattice 1 in whichthe repeating unit (which is also a unit cell) may be taken as, forexample, one of the first oligomer assemblies 3, together with and halfof each of the adjacent second oligomer assemblies 4 formed by thefurther monomers 6 fused to the first monomers 5 of that first oligomerassembly 1. Clearly visible from FIG. 1 is the symmetry of the proteinlattice 1 based on the symmetries of the first oligomer assembly 3 andthe further oligomer assembly 4. In particular as the rotationalsymmetry axes of order 4 of the further oligomer assembly 4 are alignedwith the set of rotational symmetry axes of order 4 of the firstoligomer assembly 3 the symmetry of the lattice is the same as thesymmetry of the set of rotational symmetry axes.

In the platonic class, the further oligomer assembly belongs to aplatonic point group as well as the first oligomer assembly.

In the first two protein lattices where the protomers belong to platonicpoint groups of the same order, the first and further oligomerassemblies may be identical, in which case the first and furthermonomers are also identical, or may be different oligomer assembliesbelonging to an identical point group. The set of rotational symmetryaxes of order N around which is formed an N-fold fusion are theprincipal rotational symmetry axes of the two oligomer assemblies.

In the third protein lattice in the platonic class where the first andfurther oligomer assemblies belong respectively to tetrahedral andoctahedral point groups (or vice versa), the rotational symmetry axes oforder N around which the N-fold fusion occurs are the rotationalsymmetry axes of order 3 of the two oligomer assemblies. In this case,either one of the oligomer assemblies may be considered as the firstoligomer assembly. If the oligomer assembly belonging to a tetrahedralpoint group is considered as the first oligomer assembly, then the setof rotational symmetry axes are the principal rotational symmetry axes.If the oligomer assembly belonging to an octahedral point group isconsidered as the first oligomer assembly, then the set of rotationalsymmetry axes are the set of rotational symmetry axes of order 3,because this is the order of the rotational symmetry axes of the furtheroligomer assembly belonging to the tetrahedral point group.

The platonic class may be visualised by considering each oligomerassembly as a node from which the set of rotational symmetry axes oforder N extend outwardly and joined to the rotational symmetry axes ofan oligomer assembly of the opposite type.

Lastly, in the dihedral class, the protomers comprise three monomers allbelonging to a dihedral point group. The central monomer may beconsidered as the first monomer of a first oligomer assembly belongingto a dihedral point group of order 3, 4 or 6. The monomers fused to eachterminus of the first oligomer assembly may each be considered as thefurther monomers. One of the further monomers is a monomer of a furtheroligomer assembly belonging to a dihedral point group of the same orderas the dihedral point group of the first oligomer assembly. Thus, as aresult of the symmetries of the first oligomer assembly and this one ofthe further oligomer assembly, this results of the formation of arepeating unit in which the principal rotational symmetry axes of botholigomer assemblies (i.e. the rotational symmetry axis of the same orderas the dihedral point group) are aligned. Therefore, in the proteinlattice, these oligomer assemblies are arranged in columns along whichthe first and further oligomer assemblies are alternately arranged.

The other of the further monomers is a monomer of an oligomer assemblybelonging to a dihedral point group of order 2 and so have a rotationalsymmetry axis of order 2 which is equal to the rotational symmetry axisof order 2 of the first oligomer assembly. Such rotational symmetry axesof the first oligomer assembly are equal in number to the order of thedihedral point group to which the first oligomer assembly belongs, andextend perpendicular to the principal rotational symmetry axis of thedihedral point group, being arranged symmetrically around that principalrotational symmetry axis. Therefore, the further oligomer assembliesbelonging to a dihedral point group of order 2 are arranged in theassembled protein lattice with their principal rotational symmetry axesaligned to the just described rotational symmetry axes of order 2 of thefirst oligomer assembly. As these extend perpendicular to the principalrotational symmetry axes of the first oligomer assembly, the furtheroligomer assemblies belonging to a dihedral point group of order 2 maybe considered as links between the columns of oligomer assembliesdescribed above.

In other words, the set of rotational symmetry axes of the firstoligomer assembly includes the principal rotational symmetry axis oforder 3, 4 or 6, together with the rotational symmetry axes of order 2perpendicular to the principal rotational symmetry axis.

In other classes of protein lattice, the protomers are heterologous withrespect to the monomers i.e. there are two or more types of protomer inthe protein lattice. To achieve assembly of any two types of protomer,the two types of protomer include different monomers of the sameheterologous oligomer assembly. Thus when the protomers of the differenttypes are allowed to assemble, the heterologous oligomer assembliesassemble, thereby linking the protomers of the two types. However, incontrast to homologous protomers, a single type of protomer cannot byitself assemble into the entire protein lattice. The individual monomersof the heterologous oligomer assembly cannot self-assemble into theentire heterologous oligomer assembly in the absence of the other,different monomers of that heterologous assembly. This providesadvantages during manufacture of the protein lattices, because each typeof protomer may be separately produced without assembly of an entireprotein lattice which might otherwise disrupt the production of theprotomer. This allows production in a two-stage process, which will bedescribed in more detail below.

Preferably, the heterologous oligomer assembly belongs to a cyclic pointgroup. In this case, the heterologous oligomer assembly may constitute afurther oligomer assembly which is fused in the assembled lattice by anN-fold fusion to the first oligomer assembly.

In the simplest types of protein lattice, the heterologous protomerseach further comprise a monomer of a homologous oligomer assembly, whichmay be the first oligomer assembly. The individual types of protomer mayassemble into a respective, discrete component of the unit cell, as aresult of the monomers of the homologous oligomer assemblyself-assembling. This is an advantage of the heterologous protomers,because assembly of the lattice may be avoided until the components arebrought together. Otherwise assembly of the lattice might hinder theproduction of the protomers themselves.

For example, Table 2 represents some simple heterologous protomerscapable of forming a protein lattice. TABLE 2 Heterologous ProtomersCompo- 1st Protomer 2nd Protomer Protomer nents Name M N M N p₃c_(3A) +P₃/P₃ Platonic 12 3 12 3 p₃c_(3A)* p₄c_(3A) + P₄/P₄ Platonic 24 3 12 3p₃c_(3A)* p₄c_(3A) + P₄/P₃ Platonic 24 3 12 3 p₃c_(3A)* p₃c_(3A) + P₃/D₃Mixed 12 3 d₃c_(3A)* p₃c_(2A) + P₃/D₂ Mixed 12 2 d₂c_(2A)* p₄c_(4A) +P₄/D₄ Mixed 24 4 d₄c_(4A)* p₄c_(3A) + P₄/D₃ Mixed 24 3 d₃c_(3A)*p₄c_(2A) _(3A) + P₄/D₂ Mixed 24 2 d₂c_(2A)* c_(3A)d₃d₂ + D₃/D₃ Dihedral6 3, 2 6 3, 2 c_(3A)*d₃d₂ c_(4A)d₄d₂ + D₃/D₃ Dihedral 8 4, 2 8 4, 2c_(4A)*d₄d₂ c_(6A)d₆d₂ + D₆/D₆ Dihedral 12 6, 2 12 6, 2 c_(6A)*d₆d₂d₃d₃c_(2A) + D₃/D₃ Dihedral 6 3, 2 6 3, 2 d₃d₃ c_(2A)* d₄d₄c_(2A) +D₄/D₄ Dihedral 8 4, 2 8 4, 2 d₄d₄ c_(2A)* d₆d₆c_(2A) + D₆/D₆ Dihedral 126, 2 12 6, 2 d₆d₆ c_(2A)* c_(3A)d₃c_(2B) + D₃/D₃ Dihedral 6 3, 2 6 3, 2c_(3A)* d₃c_(2B)* c_(4A)d₄c_(2B) + D₄/D₄ Dihedral 8 4, 2 8 4, 2 c_(4A)*d₄c_(2B)* c_(6A)d₆c_(2B) + D₆/D₆ Dihedral 12 6, 2 12 6, 2 c_(6A)*d₆c_(2B)*

In Table 2, monomers of a single heterologous oligomer assemblybelonging to a cyclic point group are used so that the protein latticeis formed from two types of protomer identified in the first column.Each of the protomers includes one of the monomers of the heterologousoligomer assembly.

In Table 2, the monomers of each protomer are identified by lower caseletters in similar manner as in Table 1. The lower case letters p and dhave the same meaning as in Table 1. In addition, lower case crepresents a monomer of a heterologous oligomer assembly belonging to acyclic point group. The subscript number again represents the order ofthe point group. The subscript capital letters A and A* are used toidentify the two different monomers of the same heterologous assembly.

In Table 2, the second column identifies the point groups to which thecomponents resulting from the assembly of each type of protomer belongs.A similar notation is used as for the monomers of the protomer, exceptthat capital letters are used to indicate that the point group of thecomponent is being referred to. Thus capital letter P indicates that thecomponent belongs to a platonic point group, so P₃ represents atetrahedral point group and P₄ represents an octahedral point group.Capital letter D indicates that the component belongs to a dihedralpoint group. In a similar manner to Table 1, the final columns give, inrespect of each protomer where appropriate, the number M of monomers inthe first oligomer assembly and the order(s) N of the set of rotationalsymmetry axes of the first oligomer assembly which are aligned with therotational symmetry axis of a further oligomer assembly.

For ease of reference, the protein lattices are divided into classes onthe basis of the symmetry of their components, in a similar manner tothe division of the protein lattices formed from homologous protomers.In each case, the heterologous protomers may be derived from theprotomers of the corresponding class of homologous protomer in Table 1.

For the mixed class and the platonic class, the two types of protomersboth comprise:

-   (a) a monomer of a homologous oligomer assembly which belongs to the    same point group as a respective one of the monomers of the    corresponding homologous protomer; and-   (b) a monomer which is a respective one of the two different    monomers of the heterologous oligomer assembly which belongs to a    cyclic point group.

The order of the cyclic point group to which the heterologous oligomerassembly belongs is the same as the order N of the N-fold fusion betweenthe oligomer assemblies of the protein lattice formed from thecorresponding homologous protomer, that is the order of the respectiverotational symmetry axis of the first oligomer assembly.

Thus, in the assembled protein lattice, the repeating unit hasfundamentally the same arrangement as the repeating unit of thecorresponding homologous protomer, except as follows. Instead of theN-fold fusion between the two homologous oligomer assemblies of thehomologous protomer, the link between the homologous oligomer assembliesis extended by the insertion of the heterologous oligomer assembly.Therefore, it will be seen that the repeating unit of the heterologousoligomer assembly effectively extends the length of the links of therepeating unit between the first oligomer assemblies which may beconsidered as notes in the protein lattice. Thus, the size of the poreswithin the protein lattice is also increased relative to the use of thecorresponding homologous protomers. Increasing the size of the pores inthis manner represents a significant advantage of the use ofheterologous protomers.

FIG. 2 illustrates a particular example of a protein lattice 7 belongingto the mixed class, in particular having respective protomers 8 and 9represented by p₃c_(3A) and d₃c_(3A•), respectively. The first protomer8 comprises a first monomer 10 of a first homologous oligomer assembly11, namely is E. Coli dps which belongs to a tetrahedral point group.Fused to the first monomer 10 in the first protomer 8 is a furthermonomer 12 of a further heterologous oligomer assembly 13, namelybacteriophage T4 gp5 and gp27 which belongs to a cyclic point group oforder 3. On assembly, the first protomer 8 forms a first component 14 bythe first monomers 10 assembling together. The first component 14 hasthe same symmetry as the first oligomer assembly 11 of the firstprotomer 8.

The second protomer 9 comprises a monomer 15 which is the other monomerof the further oligomer 13 of the first protomer 8 which is heterologousto the further monomer 12 of the first protomer 8. The second protomer 9also comprises a monomer 16 which is a monomer of a homologous oligomerassembly 17, namely human PTPS which belongs to a dihedral D₃ pointgroup of order 3. On assembly, the second protomer 9 forms a secondcomponent 18 by the homologous monomers 16 assembling together.

When the first and second components 14 and 18 are brought together,they assemble to form the protein lattice 7 by assembly of theheterologous oligomer assembly 13. It is clearly visible from FIG. 2 howthe symmetry of the protein lattice 7 is based on the symmetries of thehomologous oligomer assemblies 11 and 17. In particular, the rotationalsymmetry axes of order 3 of both the beterologous oligomer assembly 13and the homologous oligomer assembly 17 of the second protomer 9 arealigned with the set of rotational symmetry axes of order 3 of the firstoligomer assembly 11 of the first protomer 8. It is further clear fromFIG. 2 how the heterologous oligomer assemblies 13 effectively extendthe length of the links between the first oligomer assemblies 11. In thelattice 7, the repeating unit may be taken, for example, as one of thefirst components 14 and half of each of the adjacent second components18. In this case, the unit cell is formed by a number of such repeatingunits combined together.

The protomers of the dihedral class of the heterologous compriseprotomers comprising three monomers which may be derived from acorresponding one of the dihedral class of homologous protomers. Inparticular, the two types of protomer comprise the correspondinghomologous protomer with either one (or both) of the further monomers ofthe corresponding homologous protomers replaced by respective monomersof a heterologous oligomer assembly belonging to a cyclic point group ofthe same order as the dihedral point group to which the oligomerassembly of the replaced monomer belongs.

The above examples of protein lattices are believed to represent thesimplest form of protomers capable of forming a protein lattice and arepreferred for that reason. However, it will be appreciated that otherprotomers formed from monomers of oligomer assemblies having suitablesymmetries will be capable of forming a protein lattice. For example,other homologous protomers having larger numbers of monomers than listedin Table 1 will be capable of forming a protein lattice. Similarly,other heterologous protomers will be capable of forming a proteinlattice. These may include two types of protomer having larger numbersof monomers than in the examples of Table 2, or may include more thantwo types of protomer.

For each of the monomers, there is a large choice of oligomer assemblieshaving the required symmetry. The present invention is not limited toparticular oligomer assemblies, because in principle any oligomerassembly having a quaternary structure with the requisite symmetry maybe used. However, as examples Table 3 lists some possible choices ofoligomer assembly for each of the point groups of Tables 1 and 2. TABLE3 Example oligomer assemblies Point Name of Oligomer PDB Group SourceAssembly Code P₃(T, 32) E. coli dps 1DPS S. epidermis EpiD 1G63 P₄(O,432) Human heavy chain ferritin 2FHA E. coli Dihydrolipoamide 1E2Osuccinyltransferase A. vinelandii Dihydrolipoamide 1EABacetyltransferase D₂ Human Mn superoxide 1AP5 P. falciparum dismutaselactate dehydrogenase 1CEQ D₃ Rat 6-pyruvoyl 1B66 tetrahydropterinsynthase E. coli Amino acid 1I1L aminotransferase D₄ E. coli PurE 1QCZSipunculid worm Hemerythrin 2HMQ D₆ S. typhimurium Glutamine Synthetase1F1H C_(2A) + C_(2A)* Human Casein kinase alpha 1JWH and beta chainsC_(3A) + C_(3A)* Coliphate T4 gp5 + gp27 1K28 HIV N36 + C34 1AIKPseudomonas putida Napthalene 1NDO 1,2-Dioxygenase C_(4A) + C_(4A)*Erachiopod Hemerythrin N/A

Thus the present invention provides a protein protomer or plural proteinprotomers capable of assembly into a protein lattice. The monomers ofthe protomer may be of any length but typically have a length of 5 to1000 amino acids, preferably at least 20 amino acids and/or preferablyat most 500 amino acids.

The invention also provides polynucleotides which encode the proteinprotomers of the invention. The polynucleotide will typically alsocomprise an additional sequence beyond the 5 and/or 3 ends of the codingsequence. The polynucleotide typically has a length of at least threetimes the length of the encoded protomer. The polynucleotide may be RNAor DNA, including genomic DNA, synthetic DNA or cDNA. The polynucleotidemay be single or double stranded.

The polynucleotides may comprise synthetic or modified nucleotides, suchas methylphosphonate and phosphorothioate backbones or the addition ofacridine or polylysine chains at the 3′ and/or 5′ ends of the molecule.

Such polynucleotides may be produced and used using standard techniques.For example, the comments made in WO-00/68248 about nucleic acids andtheir uses apply equally to the polynucleotides of the presentinvention.

The monomers are typically combined to form protomers by fusion of therespective genes at the genetic level (e.g. by removing the stop codonof the 5′ gene and allowing an in-frame read through to the 3′ gene). Inthis case the recombinant gene is expressed as a single polypeptide. Thegenes may, alternatively, be fused at a position other than the endterminus so long as the quaternary structure of the oligomer assemblyproperties remains substantially unaffected. In particular, one gene maybe inserted within a structurally tolerant region of a second gene toproduce an in-frame fusion.

Chemical fusion of the polypeptide chains may be used as an alternativeto fusion at the genetic level. In this instance the polypeptides arefused post-translationally by means of the covalent linkage, but inparticular through the use of intein chemistry.

The invention also provides expression vectors which comprisepolynucleotides of the invention and which are capable of expressing aprotein protomer of the invention. Such vectors may also compriseappropriate initiators, promoters, enhancers and other elements, such asfor example polyadenylation signals which may be necessary, and whichare positioned in the correct orientation, in order to allow for proteinexpression.

Thus the coding sequence in the vector is operably linked to suchelements so that they provide for expression of the coding sequence(typically in a cell). The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner.

The vector may be for example, plasmid, virus or phage vector. Typicallythe vector has an origin of replication. The vector may comprise one ormore selectable marker genes, for example an ampicillin resistance genein the case of a bacterial plasmid or a resistance gene for a fungalvector.

Promoters and other expression regulation signals may be selected to becompatible with the host cell for which expression is designed. Forexample, yeast promoters include S. cerevisiae GALA and ADH promoters,S. pombe nmt1 and adh promoter. Mammalian promoters include themetallothionein promoter which can be induced in response to heavymetals such as cadmium. Viral promoters such as the SV40 large T antigenpromoter or adenovirus promoters may also be used.

Mammalian promoters, such as β-actin promoters, may be used.Tissue-specific promoters are especially preferred. Viral promoters mayalso be used, for example the Moloney murine leukaemia virus longterminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter,the SV40 promoter, the human cytomegalovirus (CMV) IE promoter,adenovirus, HSV promoters (such as the HSV IE promoters), or HPVpromoters, particularly the HPV upstream regulatory region (URR).

Another method that can be used for the expression of the proteinprotomers is cell-free expression, for example bacterial, yeast ormammalian.

The invention also includes cells that have been modified to express theprotomers of the invention. Such cells include transient, or preferablystable higher eukaryotic cell lines, such as mammalian cells or insectcells, using for example a baculovirus expression system, lowereukaryotic cells, such as yeast or prokaryotic cells such as bacterialcells. Particular examples of cells which may be modified by insertionof vectors encoding for a polypeptide according to the invention includemammalian HEK293T, CHO, HeLa and COS cells. Preferably the cell lineselected will be one which is not only stable, but also allows formature glycosylation of a polypeptide. Expression may be achieved intransformed oocytes.

The protein protomers, polynucleotides, vectors or cells of theinvention may be present in a substantially isolated form. They may alsobe in a substantially purified form, in which case they will generallycomprise at least 90%, e.g. at least 95%, 98% or 99%, of the proteins,polynucleotides, cells or dry mass of the preparation.

The protomers may be prepared using the vectors and host cells usingstandard techniques. For example, the comments made in WO-00/68248regarding methods of preparing protomers (referred to as “fusionproteins” in WO-00/68248) apply equally to preparation of protomersaccording to the present invention.

Assembly of the protein lattice from the protomers may be performedsimply by placing the protomers under suitable conditions forself-assembly of the monomers of the oligomer assemblies. Typically,this will be performed by placing the protomers in solution, preferablyan aqueous solution. Typically, the suitable conditions will correspondto those in which the naturally occurring protein self-assembles innature. Suitable conditions may be those specifically disclosed inWO-00/68248.

In the case of homologous protomers this results in direct assembly ofthe protein-lattice.

In the case of heterologous protomers, assembly is preferably performedin plural stages. In a first stage, each type of protomer is separatelyassembled into a respective discrete component. In a second stage, thediscrete components are brought together and assembled into the proteinlattice. Where plural heterologous protomers are used, there may befurther stages intermediate the first and second stage in which therespective discrete components are brought together and assembled intolarger, intermediate components.

A specific protein lattice of the type illustrated in FIG. 1 has beenprepared using the following method.

Human ferritin heavy chain (HFH) and the E. coli PurE gene wereamplified by PCR from human cDNA and E. coli gDNA respectively. Primersfor amplification of the ferritin gene were: 5′-CCT TAG TCG AAT TCA TGACGA CCG CGT CCA CC-3′ and 5′-GGG AAA TTA GCC CTC GAG TTA GCT TTC ATTATC-3′. Primers for amplification of the PurE gene were: 5′-GTT TTA AGACCC ATG GCT TCC CGC AAT AAT CCG-3′ and 5′-CGC AAA CCT GGA TCC TGC CGCACC TCG CGG-3′. The PurE gene was cloned into the pET-28b vector(Novagen) between the NcoI and BamHI sites. The HFH gene was cloned intothe resulting vector between the EcoRI and XhoI sites to create anin-frame fusion of the two genes under control of the T7lac promoter.

This vector was transformed into E. Coli strain B834(pLysS) forexpression. Induction of expression was as follows: a 10 ml overnightculture of the expression strain (in LB broth containing 30 μg/mlKanamycin) was diluted 1:100 into fresh LB broth containing 30 μg/mlKanamycin, Cells were grown with shaking at 37° C. to a densitycorresponding to an OD₆₀₀ of 0.6 and were then induced to express thetarget protein by the addition of IPTG to a final concentration of 1 mM.The culture was maintained at 37° C. with shaking for a further 3 hoursbefore the cells were harvested by centrifugation (5000 g, 10 min, 4°C.). The cell pellet was resuspended in 20 ml of buffer A (300 mM NaCl,1 mM EDTA, 50 mM HEPES, pH7.5). Cells were lysed by sonication and theinsoluble fraction harvested by centrifugation (25,000 g, 30 min, 4°C.). This fraction was dissolved in SM urea and centrifuged (25,000 g,30 min, 4° C.) to remove insoluble particles. The urea solubilisedmaterial was concentrated to 16 mg/ml and passed through a 0.22 μmfilter. A drop of this material (1 μl) was then directly injected into alarger drop (5 μl) of buffer A. Protein lattice particles were observedwithin one hour. FIG. 3 is a picture of one of the protein latticeparticles having a diameter of approximately 0.6 mm. The elementalcomposition of the protein lattice has been confined using μPIXEtechniques.

Protein lattices in accordance with the present invention have numerousdifferent uses. In general, such uses will take advantage of the regularrepeating structure and the pores within the lattice. Lattices inaccordance with the present invention may be designed to have pores withdimensions expected to be of the order of nanometres to hundreds ofnanometres. Lattices may be designed with an appropriate pore size for adesired use.

The highly defined, unusually sized and finely controlled pore sizes ofthe protein lattices together with the stability of their latticestructures make them ideal for applications requiring microporousmaterials with pore sizes in the range just mentioned. As one example,the lattices are expected to be useful as a filter element or molecularsieve for filtration or separation processes. In this use, the poresizes achievable and the ability to design a pore's size would beparticularly advantageous.

In another class of use, macromolecular entities would be attached tothe protein lattice. Such attachment may be done using conventionaltechniques. The macromolecular entities may be any entities of anappropriate size, for example proteins, polynucleotides ornon-biological entities. As such, the protein lattices are expected tobe useful as biological matrices for carrying macromolecular entities,for example for use in drug delivery, or for crystallizingmacromolecular entities.

Attachment of the macromolecular entities to the protein lattice may beperformed by “tagging” either or both of the protein protomers or themacromolecular entities of interest. In this context, tagging is thecovalent addition to either or both of the protein protomers or thetarget macromolecular entities, of a structure known as a tag whichforms strong interactions with a target structure. The target structuremay be a further tag attached to the other of the protein protomer ortarget macromolecular entity, or may be a part of the protein protomeror target macromolecular entity. In the case of the protein protomer, ora macromolecular entities which is a protein, this may be achieved bythe expression of a genetically modified version of the protein to carryan additional sequence of peptide elements which constitute the tag, forexample at one of its termini, or in a loop region. Alternative methodsof adding a tag include covalent modification of a protein after it hasbeen expressed, through techniques such as intein technology.

Thus to attach the macromolecular entity to the protein lattice, theprotein protomers may include, at a predetermined position in theprotomers, an affinity tag attached to the macromolecular entity ofinterest.

Alternatively, the macromolecular entity of interest may have at apredetermined position in the protomers, an affinity tag attached to amacromolecular entity.

When a component of the protein lattice is known to form stronginteractions with a known peptide sequence, that peptide sequence may beused as a tag to be added to the target macromolecular entity. Where nosuch tight binding partner is known, suitable tags may be identified bymeans of screening. The types of screening possible are phage-displaytechniques, or redundant chemical library approaches to produce a largenumber of different short (for example 3-50 amino acid) peptides. Thetightest binding peptide elements may be identified using standardtechniques, for example amplification and sequencing in the case ofphage-displayed libraries or by means of peptide sequencing in the caseof redundant libraries.

To attach the macromolecular entity to the protein lattice using anaffinity tag on the lattice or the macromolecular entity, themacromolecular entity may be allowed to diffuse into, and hence becomeattached to, a pre-formed protein lattice, for example by annealing ofthe bound macromolecular entity into their lowest energy configurationsin the protein lattice may be performed using controlled cooling in aliquid nitrogen cryostream. Alternatively, the macromolecular entitiesmay be mixed with the protomers during formation of the protein latticeto assemble with the lattice.

In another class of uses, proteins having useful properties could beincorporated as one of the protomers.

A use in which an entity is attached to the protein lattice is toperform X-ray crystallography of the macromolecular entities. In thiscase, the regular structure of the protein lattice allows themacromolecular entities to be held in an array at a predeterminedposition relative to a repeating unit, so that they are held in aregular array and in a regular orientation. X-ray crystallography isimportant in biochemical research and rational drug design.

The protein lattice having an array of macromolecular entities supportedthereof may be studied using standard x-ray crystallographic techniques.Use of the protein lattice as a support in x-ray crystallography isexpected to provide numerous and significant advantages over currenttechnology and protocol for X-ray crystallography, including thefollowing:

(1) Significantly lower amounts of macromolecule will be required(probably of order micrograms rather than milligrams). This will allowdetermination of some previously intractable targets.

(2) Use of affinity tags will allow structure determination without thetypical requirement for a number of purification steps.

(3) There will be no need to crystallize the macromolecular entity. Thisis a difficult and occasionally insurmountable step in traditional X-raystructure determination.

(4) There will be no need to obtain crystalline derivatives for eachnovel crystal structure to obtain the required phase information. Sincethe majority of scattering matter will be the known protein lattice ineach case, determination of the structure may be automated and achievedrapidly by a computer user with little or no crystallographic expertise.

(5) The complexes of a protein with chemicals (substrates/drugs) andwith other proteins can be examined without requiring entirely newcrystallization conditions.

(6) The process is expected to be extremely rapid and universallyapplicable, which will provide enormous savings in time and costs.

For use in catalysing biotransformations, enzymes may be attached to theprotein lattice, or incorporated in the protein lattice.

For use in data storage, it may be possible to attach a protein which isoptically or electronically active. One example is Bacteriorhodopsin,but many other proteins can be used in this capacity. In this case, theprotein lattice would hold the attached protein in a highly orderedarray, thereby allowing the array to be addressed. The protein latticeis expected to be able to overcome the size limitations of existingmatrices for holding proteins for use in data storage.

For use in a display, it may be possible to attach a protein which isphotoactive or fluorescent. In this case, the protein lattice would holdthe attached protein in a highly ordered array, thereby allowing thearray to be addressed for displaying an image.

For use in charge separation, a protein which is capable of carrying outa charge separation process may be attached to the protein lattice, orincorporated in the protein lattice. Then the protein may be induced tocarry out the separation, for example biochemically by a “fuel” such asATP or optically in the case of a photoactive centre such as chlorophyllor a photoactive protein such as rhodopsin. A variety of chargeseparation processes might be performed in this way, for example ionpumping or development of a photo-voltaic charge.

For use as a nanowire, a protein which is capable of electricalconduction may be attached to the protein lattice, or incorporated inthe protein lattice. Using an anisotropic protein lattice, it might beable to provide the capability of carrying current in a particulardirection.

For use as a motor, proteins which are capable of inducedexpansion/contraction may be incorporated into the protein lattice.

The protein lattices may be used as a mould. For example, silicon couldbe diffused or otherwise impregnated into the pores of the proteinlattice, thus either partially or completely filling the latticeinterstices. The protein material comprising the original lattice may,if required, then be removed, for example, through the use of ahydrolysing solution.

1. A protein lattice having a regular structure with a repeating unitrepeating in three dimensions, the repeating unit comprising proteinprotomers which each comprise at least two monomers fused together, themonomers each being monomers of a respective oligomer assembly intowhich the monomers are assembled for assembly of the protomers into thelattice, wherein the repeating unit comprises protomers comprising atleast a first monomer which is a monomer of a first oligomer assemblywhich is symmetrical in three dimensions.
 2. A protein lattice accordingto claim 1, wherein the first oligomer assembly has a set of rotationalsymmetry axes extending in three dimensions whereby said repeating unitincludes protomers with the first monomers of the protomers beingassembled into said first oligomer assembly and, in respect ofrespective ones of said set of rotational symmetry axes, with furthermonomers of the protomers fused to respective first monomers beingarranged symmetrically around said respective one of said set ofrotational symmetry axes.
 3. A protein lattice according to claim 2,wherein, in said protomers, said further monomers are monomers of afurther oligomer assembly which has a rotational symmetry axis of thesame order as the respective one of said set of rotational symmetry axesof said first oligomer assembly, whereby said repeating unit includessaid protomers with said further monomers being assembled intorespective further oligomer assemblies with said rotational symmetryaxis of each respective further oligomer assembly being aligned withsaid respective one of said set of rotational symmetry axes of saidfirst oligomer assembly.
 4. A protein lattice according to claim 1,wherein the first oligomer assembly has a set of rotational symmetryaxes extending in three dimensions, and, in said protomers, furthermonomers fused to said first monomers are monomers of respective furtheroligomer assemblies which have a rotational symmetry axis of the sameorder as a respective one of said set of rotational symmetry axes ofsaid first oligomer assembly, whereby said repeating unit includesprotomers with the first monomers of the protomers being assembled intosaid first oligomer assembly and, in respect of respective ones of saidset of rotational symmetry axes, with further monomers of the protomersfused to respective first monomers being assembled into respectivefurther oligomer assemblies with said rotational symmetry axis of saidrespective further oligomer assemblies being aligned with the respectiverotational symmetry axis of said first oligomer assembly.
 5. A proteinlattice according to claim 4, wherein the orders of the rotationalsymmetry axes of said set of rotational symmetry axes are a respectiveone of 2, 3, 4, or
 6. 6. A protein lattice according to claim 5, whereineach of said monomers of said respective oligomer assemblies either is anaturally occurring protein or is based on a naturally occurring proteinwith peptide elements being absent from, substituted in, or added to thenaturally occurring protein without substantially affecting assembly ofmonomers of said respective oligomer assembly.
 7. A protein latticeaccording to claim 6, wherein, in said protomers, said monomers arefused via a linking group.
 8. A protein lattice according to claim 7,wherein the linking group is oriented relative to the first and furthermonomers in the protomer in its normal form prior to assembly to reduceany difference in the assembled lattice in either or both of theposition and orientation of (a) the termini of said first monomers intheir arrangement in said first oligomer assembly in its natural formsymmetrically around said respective one of said set of rotationalsymmetry axes of said first oligomer assembly, and (b) the termini ofsaid further monomers in their arrangement in said further oligomerassembly in its natural form symmetrically around said rotationalsymmetry axis of said respective further oligomer assembly.
 9. A proteinlattice according to claim 8, wherein the protomers are homologous withrespect to the monomers.
 10. A protein lattice according to claim 9,wherein said first oligomer assembly belongs to either a tetrahedralpoint group or an octahedral point group.
 11. A protein latticeaccording to claim 10, wherein said further oligomer assembly belongs toa dihedral point group of the same order as the respective one of saidset of rotational symmetry axes of said first oligomer assembly.
 12. Aprotein lattice according to claim 10, wherein said further oligomerassembly belongs to either a tetrahedral point group or an octahedralpoint group.
 13. A protein lattice according to claim 9, wherein saidfirst oligomer assembly belongs to a dihedral point group of order 3, 4,or 6, and said protomers comprise at least two further monomers with afurther monomer fused to each terminus of said first monomer of saidfirst oligomer assembly.
 14. A protein lattice according to claim 13,wherein one of said further monomers is a monomer of an oligomerassembly which belongs to a dihedral point group of the same order asthe dihedral point group to which the first oligomer assembly belongs.15. A protein lattice according to claim 14, wherein the other of saidfurther monomers is a monomer of an oligomer assembly which belongs to adihedral point group of order
 2. 16. A protein lattice according toclaim 8, wherein the protomers are heterologous with respect to themonomers.
 17. A protein lattice according to claim 16, wherein the unitcell includes protein protomers of two types, wherein the two types ofprotomer include different monomers of the same heterologous oligomerassembly.
 18. A protein lattice according to claim 17, wherein at leasta first type of protomer constitutes said protomers with the firstmonomers of the protomers being assembled into said first oligomerassembly and said further monomers of the protomers fused to respectivefirst monomers are one of said different monomers of the sameheterologous oligomer assembly, said heterologous oligomer assemblybelonging to a cyclic point group.
 19. A protein lattice according toclaim 18, wherein said first oligomer assembly of the first type ofprotomer belongs to either a tetrahedral point groups or an octahedralpoint group.
 20. A protein lattice according to claim 19, wherein thesecond type of protomer comprises a monomer which is a monomer of anoligomer assembly belonging to a dihedral point group of the same orderas said heterologous oligomer assembly.
 21. A protein lattice accordingto claim 18, wherein the second type of protomer comprises a monomerwhich is a monomer of an oligomer assembly belonging to either atetrahedral point group or an octahedral point group.
 22. A proteinlattice according to claim 1 having an array of macromolecular entitiesattached thereto.
 23. A protein lattice according to claim 22, whereinthe protomers have, at a predetermined position in the protomers, anaffinity tag attached to a macromolecular entity.
 24. A protein latticeaccording to claim 23, wherein the macromolecular entities have apeptide affinity tag attached to one of the protomers in the proteinlattice.
 25. Use of a protein lattice according to claim 1 as a supportfor the array of macromolecular entities for x-ray crystallography ofthe macromolecular entities.
 26. A method of performing x-raycrystallography comprising supporting an array of macromolecularentities on a protein lattice according to claim 1 and performing x-raycrystallography on the lattice having the macromolecular entitiessupported thereon.
 27. A protein protomer comprising at least twomonomers fused together, the monomers each being monomers of arespective oligomer assembly into which the monomers are capable ofself-assembly to assemble at least part of a repeating unit of theprotein lattice having a regular structure repeating in threedimensions, wherein, in said protomer, at least a first monomer is amonomer of a first oligomer assembly which is symmetrical in threedimensions.
 28. A protein promoter according to claim 27, wherein thefirst oligomer assembly has a set of rotational symmetry axes extendingin three dimensions, and, in said protomers, further monomers fused tothe first monomers are monomers of respective further oligomerassemblies which have a rotational symmetry axis of the same order as arespective one of said set of the rotational symmetry axes of said firstoligomer assembly, whereby said repeating unit includes protomers withthe first monomers of the protomers being assembled into said firstoligomer assembly and, in respect of respective ones of said set ofrotational symmetry axes, with further monomers of the protomers fusedto respective first monomers being assembled into respective furtheroligomer assemblies with said rotational symmetry axis of saidrespective further oligomer assemblies being aligned with the respectiverotational symmetry axis of said first oligomer assembly.
 29. Pluraldifferent protein protomers according to claim 28, wherein the monomersof the plural different protomers are capable of self-assembly with eachother to form the entire protein lattice.
 30. A polynucleotide encodinga protein protomer according to claim
 28. 31. A vector capable ofexpressing a protomer according to claim
 28. 32. A host cell comprisinga vector according to claim
 31. 33. A method of making a proteinprotomer according to claim 28, comprising expressing a polynucleotidesequence which encodes the protomer in a host cell and, optionally,purifying the expressed protomer.
 34. (canceled)